JP2007501625A - Avian spermatogonial stem cell culture method and avian spermatogonial stem cell obtained thereby - Google Patents

Abstract

The present invention comprises (a) obtaining a testis of a bird; (b) separating a testis cell population from the testis; and (c) a spermatogonial stem cell contained in the testis population on a basal cell. Thus, the present invention relates to a method for long-term culture of avian spermatogonial stem cells including a step of culturing in a medium containing a cell growth factor, a population of avian spermatogonial stem cells, and a method of producing transformed birds.
[Selection] Figure 11a

Description

The present invention relates to a method for long-term culture of avian spermatogonial stem cells, a population of avian spermatogonial stem cells, and a method for producing transformed birds.

The spermatogenesis process is a process in which the spermatogonia of the male testis form sperm through the process of division, differentiation, and cell apoptosis. Therefore, the spermatogenesis process is very complex and has a systematic and efficient production system. The spermatogenesis process in chickens is very similar to that of mammals, and, like mammals, two organs, semiminiferous tubules and interstitial cells, interact in a complex manner. Become.

Avian spermatogonia are derived from primordial germ cells (PGCs), develop from epiblasts, and gradually migrate from the lower layers during the early stages of primitive streak formation It begins to gather at the germinal crescent site outside the embryo via the hypoblast. Thereafter, while the vascular system develops, primordial germ cells (PGC) circulate through the blood vessels and move to the gonads and develop into spermatogonia within the testis.

On the other hand, spermatogonial stem cells have self-renewal and the ability to produce sperm (Morrison et al., 1997), and in the case of mice, one spermatogonial stem cell becomes a spermatocyte. In other words, the cell divides about 10 times, that is, one stem cell becomes 1024 spermatocytes, and after a series of meiosis, 4096 spermatozoa are formed. Of course, 75-90% of this is lost through the cell apoptosis process (Russell et al., 1990).

There are very few spermatogonial stem cells in the testis, but in the case of mice, there are about 10 ⁸ cells in the testis, of which approximately 2 × 10 ⁴ are stem cells. It is inferred (Meistrich & Beek, 1993; Tegelenbosch & de Rooij, 1993). Among the spermatogonia, cells of interest are spermatogonial stem cells that have self-renewing ability and spermatogenic ability throughout the adult period.

There were many attempts to reproduce the spermatogenesis process in vitro using the isolated germ cells, but it did not succeed, co-cultured mouse immature germ cells with Sertoli cells, Although successfully differentiated into haploid spermatids (Rassoulzadegan et al., 1993), in vitro spermatogenesis still has technical limitations. To date, in vitro culture systems have been reported to be difficult to exceed several weeks (Ogawa, 2001; Dirmai et al., 1999; Nagano et al., 1998), and in the case of mice, about 4 months It was reported that a normal spermatogenesis process occurs after maintenance and transplantation into the receptor (Nagano et al., 1998). Therefore, the separation of spermatogonia is limited, and a large number of spermatogonia cells die during culturing, making it difficult to cultivate spermatogonia. The biggest problem is the lack of distinguishable morphological and biochemical markers (Nagano et al., 1998; van Pelt et al., 2002).

On the other hand, Shinohara et al. (1999) reported that α6-integrin and β1-integrin antibodies react with mouse spermatogonial stem cells to distinguish them from other tissues and can be used as marker markers. Then, the lectin DBA (Dolichos biflorus agglutinin) showed a specific reaction to testicular gonocytes and spermatogonia up to 30 weeks of age, and was proved to be usable as a specific marker. (Ertl and Wrobel, 1992).

Although there are no reports on spermatogonia through culturing of mammals including mice, mTERT (mouse telomerase catalytic component) (Feng et al., 2002), SV40 large T antigen (van Pelt et al., 2002), etc. were used. Establishment of mouse and rat spermatogonia cell lines has been reported.

Either testicular cells of livestock such as cattle, pigs and horses can be cultured and transplanted into mouse testis (Dobrinski et al., 2000), or cow type A spermatogonia can be cultured for a long time (about 150 days). However, there have been reports of division and differentiation aspects of spermatogonia (Izadyar et al., 2003), and in the case of humans, culturing spermatogonia is primarily intended for the treatment of diseases such as azoospermia. However, when differentiated into spermatids and fertilized, they cannot develop into morulas and have problems such as sex chromosome abnormalities (Sousa et al., 2002). ).

However, research on the culture and utilization of avian spermatogonia, including chickens, is rarely done compared to mammals, and has recently attracted attention as a tool for producing transformed birds. . Such a spermatogonia cell line is an important tool that reveals the molecular mechanism of the spermatogenesis process, and can be used for production of transformed individuals and gene therapy of germ cells through genetic manipulation and modification.

Numerous papers are referenced throughout this specification and their citations are displayed. The disclosure content of the cited paper is incorporated herein by reference in its entirety, and the level of the technical field to which the present invention belongs and the content of the present invention are explained more clearly.

As a result of diligent research to solve the above-mentioned demands in the industry, the present inventors have established a method for culturing avian spermatogonial stem cells and elucidated the characteristics of avian spermatogonial stem cells obtained through this method. The present invention has been completed.

Therefore, an object of the present invention is to provide a method for long-term culture of avian spermatogonial stem cells.

  Another object of the present invention is to provide a population of avian spermatogonial stem cells comprising avian cells exhibiting the characteristics of spermatogonial stem cells.

Still another object of the present invention is to provide a method for producing transformed birds using spermatogonial stem cells.

  Other objects and advantages of the present invention will become more apparent from the detailed description of the invention, the claims and the drawings.

According to one aspect of the present invention, the present invention includes (a) obtaining avian testis; (b) separating a testis cell population from the testis; and (c) comprising the testis cell population. A method of culturing avian spermatogonial stem cells on a basal cell in a medium containing a cell growth factor is provided.

The present invention is the first successful invention of establishing spermatogonial stem cell culture conditions and characterization in birds.

Hereinafter, the method of the present invention will be described in detail along each stage.

Step 1: Acquiring avian testis When the present invention is applied to a chicken, the chicken for spermatogonial stem cell culture is 70 weeks old after development, preferably 20 weeks old after development, more preferably, 2-10 weeks old males are used. Chicken testes are obtained by dissecting the cervical vertebrae and then dissecting them.

Step 2: Separating the testicular cell population from the testis The constricted tissue and membrane around the testis separated by the above process are removed, and the white membrane covering the testis tissue is removed. Next, the testis is finely cut using a scalpel and then decomposed by various decomposition methods, and then testis cells are separated.

As used herein, the term 'testicular cell' refers to spermatogonial stem cells, spermatogonia including all germ cells derived from spermatogonial stem cells, sertoli cells, stromal cells, and other muscle tissue It means a group of cells in testis tissue including cells and the like, and is mixed with the term 'testicular cell population'.

Testicular tissue degradation can be performed by various methods known in the art, and preferably, the step of separating testicular cells from the testis is performed using collagenase, trypsin, or a mixture thereof in the obtained testicular tissue. It is done by processing. More preferably, it is carried out by the two-stage enzyme treatment method, van Pelt (1996) method, or collagenase-trypsin treatment method described later, and most preferably, the collagenase-trypsin treatment method.

I. Two-stage enzyme treatment method This method is performed by the method of Ogawa et al. (1997) and its modified methods. The testicular tissue is added to HBSS (Hank's Balanced Salt's solution) in which collagenase type I is dissolved, and after a predetermined time of reaction, it is treated with trypsin.

B. van Pelt (1996) Method Testis tissue is degraded in DMEM medium in which collagenase type I, trypsin, hyaluronidase II, and DNase I are dissolved.

C. Collagenase-trypsin treatment method The testicular tissue is decomposed with HBSS in which collagenase type I and trypsin are dissolved, and the testicular tissue is further decomposed by pipetting.

The testicular tissue degradation product thus decomposed is filtered with a suitable cell strainer (pore diameter: about 70 μm), and testicular cells are recovered.

Step 3: Step of culturing avian spermatogonial stem cells contained in the population of testis cells The testis cells obtained by the above-described process are cultured on a basal cell in a medium containing cell growth factor, and the population of testis cells is increased. Avian spermatogonial stem cells included in the culture.

In the culture of spermatogonial stem cells, basal cells are essentially used, and spermatogonial stem cells are attached to the basal cell layer and proliferate to form colonies. According to a preferred embodiment of the present invention, the basal cell is a fibroblast, a genital matrix cell, a testicular matrix cell, or a mouse STO cell line (SIM mouse embryo-derived, Thioguanine- and Quabain-resistant fibroblast cell line). More preferred are genital matrix cells or testicular matrix cells, and most preferred are genital matrix cells. When the method of the present invention is applied to chickens, the fibroblasts, genital matrix cells, and testicular matrix cells are preferably derived from chickens.

The basal cells are located at the bottom of the dish or plate containing the medium, and the spermatogonial stem cells transferred to the medium are attached to the basal cell layer and proliferate.

On the other hand, a medium used for culturing spermatogonial stem cells contains a cell growth factor as an essential component, but preferably a fibroblast growth factor (for example, basic fibroblast growth factor), insulin-like Including insulin-like growth factor-1, stem cell factor, glial derived neurotrophic factor, or a combination thereof, more preferably fibroblasts Including growth factor, insulin-like growth factor-1, stem cell factor, or combinations thereof, most preferably a mixture of fibroblast growth factor and insulin-like growth factor-1. Preferably, the medium used in the present invention further comprises a differentiation inhibitory factor, most preferably a leukemia inhibitory factor. Therefore, the most preferred growth factor and differentiation inhibitory factor contained in the medium of the present invention is a mixture of fibroblast growth factor, insulin-like growth factor-1, and leukemia inhibitory factor.

The medium used for the culture of the present invention preferably contains avian serum (eg, chicken serum), mammalian serum (eg, fetal bovine serum), or a mixture thereof. In addition, antioxidants (e.g., β-mercaptoethanol), antibiotics-antimycotics, non-essential amino acids (e.g., arginine, asparagine, aspartic acid, glutamic acid, glycine, proline, and serine) ), Buffer (eg, Hepes buffer), or mixtures thereof.

In the culture step of the present invention, the culture temperature is most preferably about 37 ° C. Considering that the body temperature of birds is 41 ° C., the optimum culture temperature can be said to be unique.

On the other hand, prior to the above-described culturing step, primary culture of spermatogonial stem cells can be additionally performed.

Step 4: Identification of avian spermatogonial stem cells An identification step is performed in order to confirm whether or not the avian spermatogonial stem cells cultured by the above process are authentic.

The identification includes (i) PAS (Periodic Acid Shiff's) staining, (ii) STA (Sojanum tuberosum agglutinin) staining, (iii) α6-integrin antibody staining, (iv) β1-integrin antibody staining, (v) SSEA-1 Antibody staining, (vi) SSEA-3 antibody staining, (vii) SSEA-4 antibody staining, (viii) DBA (Doliclos bifflrus agglutinin) staining, or (ix) a combination of the above staining methods. Preferably, a combination of the following staining methods is performed in order to increase the reliability of identification.

(i) PAS-stained cultured spermatogonial stem cells were fixed in a fixing solution (for example, containing phosphate buffer, glutaraldehyde, formaldehyde, and MgCl ₂ ), reacted with a periodate solution, and then subjected to Schiff reagent ( Stain by reacting with Schiff's reagent. When the cytoplasm is stained deep purple, that is, when it shows a positive reaction, it can be determined as an avian spermatogonial stem cell.

(ii) STA or DBA-stained spermatogonial stem cells are treated with a fixing solution and fixed, and then one of the lectins STA (Solanum tubersum agglutinin) or DBA (Doliclos bifflrus agglutinin) is used with a fluorescent substance (for example, FITC (fluorescein isothiocyanate)) is reacted with FITC-STA or FITC-DBA. Then, it observes with a fluorescence microscope. When fluorescence is observed on the cell surface, that is, when a positive reaction is shown, it can be determined as an avian spermatogonial stem cell.

(iii) α6-integrin antibody-stained secondary antibody (antibody Fc) obtained by treating α6-integrin antibody, which is a primary antibody, with spermatogonial stem cells and conjugated with a labeling substance such as a fluorescent substance (TRITC (tetramethyl rhodamine isothiocyanate)) An antibody that binds to a domain, such as goat anti-mouse IgG), and then observed with a fluorescence microscope. When fluorescence is observed on the cell surface, that is, when a positive reaction is shown, it can be determined as an avian spermatogonial stem cell.

(iv) β1-integrin antibody staining The same procedure as in the α6-integrin antibody method described above, except that a β1-integrin antibody is used as the primary antibody.

(v) SSEA-1, SSEA-3, or SSEA-4 antibody-stained spermatogonial stem cells are treated with a primary antibody, SSEA-1, SSEA-3, or SSEA-4 antibody, and a chromogenic reaction catalyst (for example, alkaline phosphatase) conjugated secondary antibody is processed. Next, the substrate of the catalyst is added and reacted to observe the color development aspect. When a color reaction is observed, that is, when a positive reaction is shown, it can be determined as an avian spermatogonial stem cell.

The method of the invention can be applied to a variety of birds, preferably chickens, sharks, turkeys, duck, eagle birds, moths or pigeons, most preferably chickens.

According to the method of the present invention, avian spermatogonial stem cells can be cultured for at least 2 months, preferably at least 3 months, more preferably at least 4 months, and most preferably at least 5 months.

When following the culture method of the present invention, avian spermatogonial stem cells can be obtained with high reliability. Thus, according to another aspect of the present invention, the present invention provides a population of avian spermatogonial stem cells comprising avian cells exhibiting the characteristics of spermatogonial stem cells.

As used herein, the term 'avian spermatogonial stem cell population' means a population of cells that are composed essentially of avian spermatogonial stem cells. That is, the population of avian spermatogonial stem cells of the present invention is not only a cell population composed entirely of avian spermatogonial stem cells, but also a cell population containing trace amounts of other cells such as spermatogonia. Including.

The characteristics of the spermatogonial stem cells are (i) PAS (Periodic Acid Shiff's) staining, (ii) STA (Sojanum tuberosum agglutinin) staining, (iii) α6-integrin antibody staining, (iv) β1-integrin antibody staining, (v ) SSEA-1 antibody staining, (vi) SSEA-3 antibody staining, (vii) SSEA-4 antibody staining, (viii) DBA (Doliclos bifflrus agglutinin) staining, or (ix) a positive reaction in combination of the staining methods Means that.

According to still another aspect of the present invention, the present invention provides: (a) transferring a foreign gene to the avian spermatogonial stem cell population of the present invention; (b) accepting the avian spermatogonial stem cell population as a receptor. And (c) obtaining a progeny of the receptor to produce a transformed bird, and a method for producing a transformed bird.

In the method of the present invention, transferring a foreign gene to an avian spermatogonial stem cell can be performed by a gene transfer method commonly known in the art. For example, electroporation, liposome-mediated transfer methods (Wong et al., 1980), and retrovirus-mediated transfer methods (Chen et al., 1990; Kopchick et al., 1991; Lee & Shuman, 1990). The electroporation is most preferably performed by a method developed by the present inventors (see: Korean Patent No. 305715).

According to a preferred embodiment of the present invention, the foreign gene includes an antibiotic resistance gene as a selection marker, and after the step (a), further includes a step of selecting a spermatogonial stem cell exhibiting antibiotic resistance, The step (b) is performed with spermatogonial stem cells exhibiting antibiotic resistance. The selection marker that can be used in the present invention may be any gene that confers antibiotics to eukaryotic cells, and includes, for example, neomycin, puromycin, and zeomycin resistance genes.

In the step of transplanting the avian spermatogonial stem cells into the testis of the receptor, it is preferable to finely inject the spermatogonial stem cells into the testicular tubule.

Subsequently, the progeny is obtained by crossing the receptor with another individual, and the offspring containing the foreign gene becomes a transformed bird.

The present invention provides a method for long-term culture of avian spermatogonial stem cells, population of avian spermatogonial stem cells, and a method of producing transformed birds. When following the method of the present invention, avian spermatogonial stem cells can be obtained with reliability. The obtained avian spermatogonial stem cells can be used for molecular genetic understanding of the spermatogenesis process and production of transformed birds by genetic manipulation.

Hereinafter, the present invention will be described in more detail through examples. These examples are for explaining the present invention more specifically, and it is obvious to those skilled in the art to which the present invention belongs that the scope of the present invention is not limited to these examples. That would be true.

[Experimental materials and methods]
1) As a chicken for culturing donor chickens and testicular isolated spermatogonial stem cells, male white leghorn breeds bred at Abicore Biotechnology Laboratory Co., Ltd. were used. The chicken testis was obtained by dissecting the donor cervical vertebrae and then incising them, and measuring the body weight of each chicken by age and the weight of the testis.

2) Comparison of testicular tissue decomposition methods The isolated testis removed the tightening tissue and membranes around the testis, and the white membrane (tunica albuginea) covering the testis tissue was removed using fine tweezers. The testis was cut into pieces using a dissecting knife under a stereomicroscope, and then subjected to comparative analysis using various decomposition methods.

I. Separation method by two-step enzymatic digestion This method was performed by slightly modifying the method of Ogawa et al. (1997). That is, the testicular tissue prepared as described above was dissolved in collagenase type I (1 mg / ml, Sigma) in HBSS (Hank's Balanced Salt's solution, Invitrogen) and then shaken in a 37 ° C. shaking incubator for 15 minutes. Processed. After washing with HBSS, it was treated with 0.25% trypsin-1 mM EDTA (Invitrogen) for 15 minutes. The degraded testis cells were filtered with a 70 μm cell strainer (cell strainer, Falcon 2350), and then viability and cell number were measured using trypan blue.

B. Isolation by van Pelt (1996) method DMEM (Invitrogen) medium with collagenase type I (1 mg / ml, Sigma), trypsin (1 mg / ml, Sigma), hyaluronidase II (1 mg / ml, Sigma) and DNase I (5 μg / ml, BMS), and then the minced testis tissue was degraded in the medium at 150 cycles / min for 15 minutes. Then, after washing 3 times with DMEM medium, the second degradation was dissolved in collagenase type I (1 mg / ml), hyaluronidase II (1 mg / ml, Sigma) and DNase I (5 μg / ml, BMS). The testicular tissue was completely decomposed in DMEM medium for about 30 minutes. Then, after filtering with a 70 μm cell filter, the survival rate and the number of cells were measured.

C. Collagenase-trypsin treatment method Cells were separated in a tissue degradation medium in which collagenase type I (1 mg / ml, Sigma) and 0.25% trypsin (Sigma) were dissolved in HBSS (Invitrogen). The testis tissue was decomposed by pipetting at 5 minute intervals while decomposing the tissue at 150 rpm for 30 minutes in a 37 ° C. shaking incubator. In order to stop the activity of the enzyme, 10% FCS (fetal calf serum) was added, and then the degraded cells were collected using a cell strainer (cell strainer, 70 μm, Falcon 2350). After centrifuging at 300 × g for 5 minutes to secure testicular cells, the survival rate and the number of cells were measured using trypan blue.

3) Distribution of spermatogonial stem cells in testis tissue In order to observe the morphology of testis tissue and the distribution of spermatogonial stem cells by age of chicken, tissue analysis was performed to analyze the characteristics of testicular tissue, and STA (Solanum tuberosum agglutinin) was used to measure the number of spermatogonial stem cells.

The number of spermatogonial stem cells in testicular tissue of birds such as chickens has not yet been reported. In order to determine the number of spermatogonial stem cells, the testis of a white Leghorn species of about 3 weeks of age was degraded with collagenase-trypsin, and then the testicular cells were digested with 0.5% paraformaldehyde for about 5 minutes. Fixed. By using FITC-conjugated STA (Solanum tuberosum agglutinin, Sigma), which is one of lectins specific to spermatogonial stem cells, the cells were reacted with testis cells. After reacting for 2 hours, cells that react with STA to emit fluorescence were measured, and the distribution of spermatogonial stem cells occupying in the whole testis cells was calculated.

4) Comparison of basal cells Testicular cells initially cultured must be cultured for about 7-10 days and then transferred to an appropriate basal cell (feeder layer) for culturing. Comparative study of various basal cells. First, for the initial culture of testicular cells, testicular tissue was separated from 2-4 week-old male chicks, and then testicular tissue was decomposed by the collagenase-trypsin treatment method described above. After measuring the number of cells and the survival rate, the degraded testis tissue was inoculated with 2 × 10 ⁶ testis cells per culture dish (100 mm) and cultured for 8 to 10 days. At this time, the composition of the medium is the same as the most preferable medium of the following spermatogonial stem cells, except that basal cells are not included.

In 6-well plates (TPP, EU), chicken fibroblasts (CEF, chicken embryonic fibroblast), chicken genital matrix cells (GSC, gonadal stroma cells), or chicken testicular matrix cells (TSC, testicular stroma) to be tested as basal cells cell) was cultured (6-8 × 10 ⁴ / well). A mouse STO cell line (ATCC CRL-1503) was used after treating with mitomycin C (10 μg / ml) to suppress cell division. The spermatogonial stem cells initially cultured by the above process were cultured on basal cells prepared at 1 × 10 ⁵ cells per well for 8-10 days at 37 ° C. in a 5% CO ₂ incubator, and the number of cells was measured. And statistical processing.

5) Establishment of culture conditions based on culture solution composition To establish culture conditions based on the culture solution composition of chicken spermatogonial stem cells, the culture aspects of spermatogonial stem cells were compared using the following culture solution composition.

(i) DMEM-B (basic culture solution)
Add 10% (v / v) fetal bovine serum for ES cells (FBS, Hyclone, Logan UT) and 1x antibiotic-antimycobacterial agent (Invitrogen) to DMEM (Invitrogen) medium for basic culture composition The medium was then composed.

(ii) DMEM-C (additive)
2% chicken serum (Invitrogen), 10 mM non-essential amino acid (Invitrogen), 10 mM Hepes buffer (Invitrogen), and 0.55 mM β-mercaptoethanol (Invitrogen) are added to the above basic culture medium DMEM-B medium and cultured. The liquid was composed.

(iii) The spermatogonial stem cells were prepared by using passage 1 cells (1 × 10 ⁴ / well) and GSC basal cells (8 × 10 ³ / well) in a 24-well plate in a 5% CO ₂ incubator. After co-cultured 5 times at 37 ° C. per day, the number of colonies was measured.

6) Establishing optimal culture conditions for additives To establish optimal culture conditions for spermatogonial stem cells by comparing the culture aspects of spermatogonial stem cells for each growth factor, As a control, the culture patterns for each growth factor were compared.

(i) DMEM-C (control group) culture solution has the same culture solution composition as used above, and each growth factor, ie, 10 ng / ml human leukemia inhibitory factor (Sigma), 10 ng / ml Human basic fibroblast growth factor (Sigma), 100 ng / ml human insulin-like growth factor-1 (sigma), 20 ng / ml human stem cell factor (Sigma), and 100 ng / ml human glial-derived nerve Nutritional factors (R & D system, USA) were added and cultured at 37 ° C. in a 5% CO ₂ incubator.

(ii) For spermatogonial stem cells, use Passage 1 cells in a 24-well plate (1 × 10 ⁴ / well) and GSC basal cells (8 × 10 ³ / well) and repeat three times for 9 days. After culturing, the number of colonies was measured.

7) Effect of culture temperature on spermatogonial stem cell culture In order to establish optimal culture temperature conditions for chicken spermatogonial stem cell culture, the body temperature of birds is 41 ° C and the general culture temperature is 37 ° C. The temperature effect was compared. The culture medium was SSC medium, ie, DMEM-C (control group) culture medium with each growth factor, ie, 2 ng / ml human leukemia inhibitory factor (Sigma), 5 ng / ml human basic fibroblast growth factor (Sigma). ) And 10 ng / ml human insulin-like growth factor-1 (Sigma), and cultured in a 5% CO ₂ incubator. Testicular cells of 3-week-old white leghorn were initially cultured and cultured for 10 days, and then spermatogonial stem cells were collected and the number of cells was measured.

8) Growth curve of chicken spermatogonial stem cells For in vitro culture of chicken spermatogonial stem cells, using established culture temperature, culture medium, and basal cells, culturing after initial testicular degradation, depending on the number of culture days Changes in the number of spermatogonial stem cells were measured. That is, after initial degradation, about 2.0 × 10 ⁶ / dish (100 mm) testicular stem cells are subcultured at intervals of about 10 days while being cultured in spermatogonia culture medium and GSC basal cells. The number of spermatogonial stem cells was measured.

9) Characterization of spermatogonial stem cells using immunocytochemical methods To clarify the characteristics of chicken spermatogonial stem cells cultured from testicular tissue, PAS (Periodic Acid Schiff's) staining kit (Sigma), STA (Sigma) The appearance of spermatogonial stem cells cultured using chicken anti-integrin β1 antibody (Sigma) and chicken anti-integrin α6 antibody (Chemicon International. Inc, USA) was observed.

(i) The spermatogonial stem cells cultured by staining with PAS (Periodic Acid Shiff's) were fixed in a fixative solution (50 mM phosphate buffer, 2% glutaraldehyde, 2% formaldehyde, and 2 mM MgCl ₂ ) for 10 minutes, and then 3 times with PBS. Washed twice. After reacting with the periodic acid solution for 5 minutes, it was washed again with PBS three times. A Schiff's reagent (Sigma) was placed for 10 to 15 minutes, washed with PBS, and then observed with a microscope.

(ii) STA (Sojanum tuberosum agglutinin) -stained spermatogonial stem cells were treated with a fixative and fixed, and then FITC-STA (Sigma), one of the lectins, was diluted to 10 to 2 to 50 μg / ml. Then, the reaction was performed at room temperature for about 1 hour. Subsequently, it was washed with PBS three times and observed with a fluorescence microscope (Nikon TE2000-U, Japan).

(iii) α6-integrin and β1-integrin stained spermatogonial stem cells were treated with a fixative, washed with PBS, and cultured at room temperature for about 1 hour in order to block with 2% normal goat serum. The primary antibodies α6-integrin (Chemicon Int.) And β1-integrin antibody (Sigma) were each diluted to a concentration of 20 μg / ml and reacted at room temperature for 1 hour. As a secondary antibody, goat anti-mouse IgG (Jackson Lab) with TRITC (tetramethyl rhodamine isothiocyanate) was used. After culturing at room temperature for 1 hour, it was observed with a fluorescence microscope.

(iv) SSEA-1, SSEA-3, and SSEA-4 stained spermatogonial stem cells were treated with the fixative solution, washed with PBS, and treated with levamisole (Levamisole). In order to minimize non-specific binding of the secondary antibody, it was blocked with 5% goat serum for 30 minutes at room temperature. Next, anti-SSEA-1 (MC-480) or anti-SSEA-4 diluted 1: 100, anti-SSEA-3 (MC-631) (MC-813 diluted 1: 200) -70; Developmental Studies Hybridoma Bank, Iowa, IA) and treated for 1 hour at room temperature. The secondary antibody goat anti-mouse IgM-AP (AK-5010, Vector Laboratories, Inc., Burlingama, Calif.) Was then treated. Thereafter, the sample was reacted with ABC solution and BCIP / NBT (Sigma) substrate for 30 minutes, respectively, and 10 mM EDTA (pH 8.0) was added to stop the reaction.

(v) Double immunostained spermatogonial stem cells treated with anti-SSEA-1, anti-SSEA-3, or anti-SSEA-4, followed by rhodamine (TRITC) -conjugated goat anti-mouse IgG ( 115-025-003, Jackson ImmunoResearch Laboratories. Inc, Bar Harbor, ME). Next, after washing with PBS three times, FITC-STA was treated for 1 hour and observed with a fluorescence microscope.

[Experimental result]
1) Comparison of testicular cell separation method The enzyme treatment method for degrading chicken testis tissue was decomposed mainly with collagenase and trypsin, but the two-stage enzyme treatment method (Ogawa et al., 1997) and van Pelt (1996). ) And 3 methods of mixing collagenase and trypsin. The isolated testis cells are composed of germ cells and other somatic cells, and the cell viability was measured using trypan blue (see: Table 1 and FIG. 1). As a result of comparison of the three testicular cell separation methods, the treatment 3 method, that is, the separation method using collagenase (1 mg / ml) and trypsin (0.25%) showed the highest cell viability. Compared to the van Pelt method, it was simpler and less time consuming, so the method of treatment 3 was found to be the most efficient method for chicken testicular tissue degradation.

[Table 1]
Testicular cell viability and cell count by chicken testicular tissue degradation method

2) Distribution of spermatogonial stem cells in testis tissue There are no reports on the number of spermatogonial stem cells in the testes of birds, including chickens, but it is known that there are very few. In the case of mice, there are about 10 ⁸ cells in the testis, of which about 2 × 10 ⁴ are inferred to be stem cells (Meistrich & Beek, 1993; Tegelenbosch & de Rooij, 1993). In order to measure the number of spermatogonial stem cells in the testicular tissue of chickens, after degrading the testis of a white leghorn species of about 3 weeks of age, the STA-FITC conjugate, which is one of the lectins, is used for fluorescence. And the distribution of spermatogonial stem cells occupying in the whole testis cells was calculated (see Table 2).

Similar to mammals, birds such as chickens do not have reliable morphological and molecular chemical markers that can differentiate spermatogonial stem cells, and therefore use various types of lectins (STA, WGA, DBA, ConA). As a result, it was found that FITC-STA (Sojanum tuberosum agglutinin) specifically reacts with spermatogonial stem cells. Therefore, as a result of measurement using STA, it is inferred that about 0.8% are spermatogonial stem cells in the case of chickens. Although there are differences depending on the breed and age, in the case of 2-3 weeks old white leghorn species, the total number of cells in the testis is about 10 ⁷ , of which about 8 × 10 ⁴ are stem cells. To be judged. This is about 40 times higher than 0.02% of mice, and such a large number of spermatogonial stem cells are used for in vitro culture of chicken spermatogonial stem cells, establishment of cell lines, genetic manipulation, etc. Can be used very useful.

[Table 2]
Number of spermatogonial stem cells that specifically react with FITC-STA in testis cells

3) Establishment of optimal basal cells for spermatogonia culture There have been no reports on the culture of avian spermatogonial stem cells, including chickens, and the culture conditions for spermatogonia such as mice And tried different ways. Basically, since spermatogonial stem cells are PGC-derived cells, the EG medium is partially modified to be used as a medium (Park et al., 2000), PGC, Embryonic germ cell, and Since spermatogonia are also basal cell-dependent, chicken fibroblasts (CEF), chicken genital matrix cells (GSC), chicken testis matrix cells (TSC), and mice to find the optimal basal cells Comparison was made using an STO cell line or the like (see FIG. 2).

After the primary culture, primary cultured spermatogonial stem cells were cultured together with basal cells, and the number of spermatogonial stem cells was measured and compared. As a result of the measurement, there was no statistically significant difference from TSC, but the number of spermatogonial stem cells cultured with GSC basal cells was the highest, and the lowest values were shown for CEF and STO cell lines. Therefore, the result was obtained that co-culture of GSC as basal cells was most suitable for spermatogonial stem cell culture. That is, culturing with Sertoli cells, which occupy a large part of testis cells, can serve as nurse cells that supply growth factors necessary for proliferation and development of spermatogonial stem cells (Sousa) 2002; van der Wee et al., 2001; Rassoulzadegan et al., 1993), induces a special function of Sertoli cells, ie differentiation of spermatogonial stem cells, when co-cultured with cell lines derived from Sertoli cells such as TM4 and SF7 On the contrary, it has been found that there is a risk that the viability of spermatogonial stem cells may be reduced compared with the case of culturing with other cell lines (Nagano et al., 2003).

In the case of TSC, cells isolated from testicular tissue of chicks of 3 weeks of age or less are most preferable, and CEF tends to grow too quickly and winds, and when separating colonies, there is a disadvantage that CEF is separated together. It was. Although STO is an excellent basal cell of mouse and mammalian stem cells, colony formation is inferior to that of other basal cells when treated with mitomycin-C, and the cells continue to separate little by little. Indicated.

4) Establishment of culture conditions based on the culture medium composition To establish the culture conditions based on the culture medium composition of chicken spermatogonial stem cells, the basic medium (DMEM-B) and the medium containing additives (DMEM-C) are 9 After culturing for a day, the number of colonies was measured and compared. As a result of the measurement, colonization of chicken spermatogonial stem cells appeared about 14 times higher in DMEM-C than in DMEM-B (see: Table 3 and FIG. 3).

This is considered to be the result of additives added to the DMEM-C culture medium, chicken serum, non-essential amino acids that are metabolic related substances, Hepes buffer that is a buffer, and β- that is an antioxidant. It is judged that mercaptoethanol and the like acted in a complex manner. On the other hand, in a paper published by Nagano et al. (2003), it is reported that there is no difference in the culture of mouse spermatogonial stem cells in a basic medium and a medium to which a metabolic substrate and a buffering agent are added. The difference in the effect between the two media was large.

In the case of DMEM-B, most of the cells remained in one cell state, and the cell state showed a pattern in which the cell size was reduced (a and b in FIG. 4). On the other hand, in the case of cells grown in DMEM-C, colony formation was vigorous, and the same size and morphology as the spermatogonial stem cells at P0 were shown (c and d in FIG. 4).

[Table 3]
Comparison of number of colonies by culture solution composition

5) Establishing optimal culture conditions for additives In order to establish optimal culture conditions for spermatogonia by comparing the culture aspects of chicken spermatogonial stem cells for each growth factor, culture additives were included The culture aspects for each growth factor were compared using the culture solution as a control. Stem cell factor (SCF), leukemia inhibitory factor (LIF), and basic fibroblast growth factor (bFGF) have already been reported to promote PGC maintenance and proliferation (Matsui et al., 1992; Resnick 1992), GDNF has also been demonstrated to be an important factor regulating spermatogonial stem cell differentiation in vivo (Meng et al., 2000; Nagano et al., 2003).

In order to observe the effect on each growth factor, after culturing in a 24-well plate for about 9 days, the number of colonies was measured and compared.
(i) DMEM-C (control group)
(ii) DMEM-C + LIF (10 ng / ml)
(iii) DMEM-C + bFGF (10 ng / ml)
(iv) DMEM-C + SCF (20 ng / ml)
(v) DMEM-C + IGF-1 (100 ng / ml)
(vi) DMEM-C + GDNF (100 ng / ml)

As a result of measuring the spermatogonial stem cell colony of each well, the culture medium of the control group not containing the growth factor was rejected, and more colonies were formed than those added with LIF, bFGF, IGF-1, and GDNF, It was observed that the most colonies were formed with the addition of SCF (see Table 4 and FIG. 5). It is judged that SCF had no effect rather than stimulating spermatogonial stem cell division (Ohta et al., 2000). It was found that the addition of LIF, bFGF, or IGF-1 did not affect spermatogonial stem cell division or growth when compared with those cultured without growth factors. It can be seen that such results are somewhat consistent with those in mouse spermatogonial stem cells (Nagano et al., 2003). On the other hand, GDNF is known to cause the accumulation of undifferentiated spermatogonial stem cells by suppressing the differentiation of spermatogonial stem cells (Meng et al., 2000), and other growth factors in mouse spermatogonial stem cells. On the other hand, it appeared significantly higher, but in this experiment, the lowest result was shown (see Table 4 and FIG. 5). Looking at the culture pattern for each growth factor, a large difference is not found, but in the case of the control group, the formation and number of colonies are good, but the spermatogonia added with LIF, IGF-1, and GDNF are colonies. Formation is poor and the number is also inferior to the control.

[Table 4]
Comparison of the number of spermatogonial stem cell colonies by growth factors

Each growth factor interacts with other factors to induce a synergistic effect, but in the case of LIF, it is a necessary element for long-term culture of avian embryonic stem cells, primordial germ cells, and germ germ cells, and bFGF High effects can be expected when cultured with SCF, etc. (Pain et al., 1996; Park et al., 2000). As can be seen from this experiment, the effect on each growth factor was lower than that of the control group (DMEM-C) except for SCF (FIG. 5), but LIF, bFGF, IGF-1 and SCF were all together. When added and cultured, on the contrary, it showed a significant increase tendency higher than the control group (SCF not added: 2.8 times, SCF added: 2.2 times), and the overall effect between growth factors was observed, In the case of chicken spermatogonial stem cells, the results were lower when SCF was added compared to the treatment group that was not added, and it is judged that SCF induces differentiation or cell apoptosis of chicken spermatogonial stem cells (Table 5, (Fig. 6).

[Table 5]
Culture of chicken spermatogonial stem cells by combination of growth factors

6) Effect of culture temperature on spermatogonial stem cell culture Unlike birds, birds, including chickens, have a high body temperature (41 ° C) and testis in the body. Therefore, the testicular cells were cultured at a normal cell culture temperature of 37 ° C. and the chicken body temperature of 41 ° C., and the change in the number of spermatogonial stem cells was observed. It was confirmed that the cells were cultured 2.2 times better (refer to Table 6, FIG. 7). This is in contrast to the absence of a significant difference between 37 ° C. and the optimal in vitro testis temperature of 32 ° C. for mice (Nagano et al., 2003).

[Table 6]
Number of chicken spermatogonial stem cells by culture temperature

7) Growth curve of chicken spermatogonial stem cells Mammalian spermatogonia, including mice, can only isolate a limited number of cells during isolation, and many spermatogonia die during in vitro culture. It is difficult to culture spermatogonia. Avian spermatogonial stem cells, including chickens, show similar aspects, but have the advantage of being able to isolate more spermatogonial stem cells than mice. Because chicken spermatogonial stem cells are basal cell-dependent, they are cultured with genital matrix cells (GSC) and there are a relatively higher number of spermatogonial stem cells than in mice (approximately 0.08%). Even if a small number of spermatogonial stem cells died during the culture, the number of cells could be measured.

As a result of subculturing while culturing at intervals of about 10 days, it was found that the number of cells gradually increased until the third passage, but after that, a large number of spermatogonial stem cells died (see: Fig. 8). Actually, after Passage 4, a large number of spermatogonial stem cells went through a cell killing process, so that the total cell number decreased and only a part of spermatogonial stem cells showed a phenomenon of continuous division.

8) Establishment of long-term culture conditions for avian spermatogonial stem cells There has been no research on the culture of avian spermatogonial stem cells, especially chickens, especially long-term culture, but only mice (Nagano et al., 2001; Kanatsu- All reports have been that spermatogonial stem cells such as Shinohara et al., 2003) and cattle (Izadyar et al., 2003) have been cultured for about 5 months. On the other hand, most spermatogonial stem cells are difficult to culture because many numbers die at the initial stage of culture.

For chicken spermatogonial stem cell culture, after initial culture of whole testis cells in a culture dish for about 10 days (see: a and b in FIG. 9), colony-formed spermatogonial stem cells were isolated, A method of culturing in a culture dish prepared with basal cells was adopted (c and d in FIG. 9). In the early stage of culture, Sertoli cells and the like started to grow first, and formed a small colony composed of 3 to 4 cells (Fig. 9b), but the spermatogonial stem cells were recovered and genital basal cells When cultured with (GSC), the number of spermatogonial stem cells increased rapidly (Fig. 9c). Further, it was confirmed that growth was possible while forming colonies after passage, and that in vitro culture for a long period of 3 months or longer was possible (d in FIG. 9). Therefore, although there is a slight difference in the culture period or spermatogonia state depending on the age of the basal cells and the chickens to be separated, the present invention enables long-term culture for about 5 months. That is, spermatogonial stem cells isolated from 2-4 weeks old chickens are different from spermatogonial stem cells isolated from 5-8 weeks old and the culture of the cells is different and long-term culture is difficult. It is judged to be related to the differentiation into type B spermatogonia that occurs thereafter.

9) Because there is no reliable morphological or molecular chemical marker that can distinguish type A spermatogonial stem cells from mice and rats, including mammalian chicks with the characteristics of spermatogonial stem cells using immunocytochemical methods. Various studies have been attempted. In mice, there have been reports on α6-integrin and β1-integrin surface markers specific to gonocytes and spermatogonia (Shinohara et al., 1999), based on which chicken α6-integrin and Using the antibody against β1-integrin, PAS staining, and STA, which is a lectin, the reaction mode was observed using testis cells that were initially separated and cultured spermatogonial stem cells.

(i) PAS-stained chicken primordial germ cells (PGC) and germ germ cells (EG cells) are stained in dark purple due to the large amount of glycogen present in the cytoplasm, and can be distinguished from other cells. (Meyer, 1964; Park et al., 2000). In particular, germ germ cells tend to be specifically stained for PAS after long-term culture.

Although not at the same developmental stage, chicken spermatogonial stem cells are also derived from primordial germ cells, and as a result of staining using the PAS kit, they were stained in a deep purple color like PGC and EG cells. In particular, it was isolated and passaged from different testes at 4 weeks and 9 weeks of age, and after passage, it showed a specific staining with spermatogonial stem cells, distinguishing from other Sertoli cells and basal cells (See: FIG. 10).

(ii) STA-FITC and DBA-FITC staining reaction In order to clarify the specific reaction mode of chicken spermatogonial stem cells to lectins, DBA (Doliclos bifflrus agglutinin), STA (Solanum tubersum agglutinin) WGA (Triticum vulgaris agglutinin) As a result of using lectins such as ConA (Canavalia ensiformis agglutinin), WGA reacted to spermatogonial stem cells and basal cells, and ConA most reacted to basal cells. In particular, STA reacted specifically to spermatogonial stem cells but not to basal cells, but even after long-term culture (passage 8), it showed the same staining appearance and can be used as a specific marker for spermatogonial stem cells. (Ref: FIG. 11a). Such a result means that (N-acetylglucosamine) 3 recognized by STA is specifically present in chicken spermatogonial stem cells. DBA also reacted specifically with spermatogonial stem cells, but even after long-term culture (passage 3), it shows the same staining appearance and is judged to be usable as a specific marker for spermatogonial stem cells (see : FIG. 11b).

On the other hand, Izadyar et al. (2002) demonstrated that DBA, which is the same lectin, specifically reacts with bovine spermatogonial stem cells and can be used as a species-specific marker after pure water isolation and xenotransplantation of spermatogonial stem cells. Thus, from the results of this experiment, it is expected that STA and DBA lectin can be used as a species discrimination marker after pure water separation and xenotransplantation of chicken spermatogonial stem cells.

(iii) α6-integrin and β1-integrin reaction of chicken spermatogonial stem cells α6-integrin and β1-integrin form heterodimers in general cells and are responsible for central functions in signal transduction inside and outside the cell. In particular, α6-integrin and β1-integrin can be used as specific markers expressed from the surface of mouse spermatogonial stem cells (Shinohara et al., 1999).

On the other hand, as a result of applying chicken α6-integrin and β1-integrin to spermatogonial stem cells, it showed specificity also in chicken spermatogonial stem cells, indicating the possibility as a marker of spermatogonial stem cells (see: FIG. 12 and 13). In particular, α6-integrin showed a more specific staining on the cell surface than β1-integrin. The spermatogonial stem cell's cell membrane permeation proteins α6-integrin and β1-integrin are influenced by growth factors or inhibitors secreted from basal cells, and thus, general signaling of spermatogonial stem cells, ie differentiation or cells It is thought to affect the signal transmission of death.

(iv) SSEA-1, SSEA-3, and SSEA-4 staining SSEA-1, SSEA-3, and SSEA-4 are already known as markers for mouse spermatogonial stem cells, but for chicken spermatogonial stem cells Its use as a specific marker is not known. As can be seen from FIG. 14, chicken spermatogonial stem cells were observed to express SSEA-1, SSEA-3, and SSEA-4, and TSC (testicular stromal cell) was anti-SSEA-1, anti- No immunostaining was observed with SSEA-3 and anti-SSEA-4. Therefore, it can be seen that SSEA-1, SSEA-3, and SSEA-4 can be used as specific markers for chicken spermatogonial stem cells.

(v) Double immunostaining As can be seen from FIG. 15, chicken spermatogonial stem cells were double-stained with anti-SSEA-1 and FITC-STA.

The cell established as a chicken spermatogonial stem cell by the above-described culture process and identification process is named chSSC, deposited with the Korean Cell Line Research Foundation on June 14, 2003, and assigned the deposit number KCLRF-BP-08080 It was done.

Although specific portions of the present invention have been described in detail above, such specific descriptions are merely preferred embodiments for those having ordinary skill in the art, and the scope of the present invention is limited thereto. Obviously, the substantial scope of the present invention is defined by the appended claims and their equivalents.

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It is a graph which shows the cell survival rate by the decomposition | disassembly method conditions of a chicken testis tissue, Comprising: The process 1 is a two-step enzyme treatment method, The process 2 is the van Pelt (1996) method, The process 3 is a collagenase-trypsin treatment method. . It is a graph which shows the culture | cultivation aspect of the chicken spermatogonial stem cell by a basal cell. After the cultivated spermatogonial stem cells were co-cultured with the respective basal cells, the number of spermatogonial stem cells was observed and compared on the 8th day. It is the graph which compared the number of colonies of the chicken spermatogonial stem cell by a culture solution composition. It is a photograph which shows the culture | cultivation form of the chicken spermatogonial stem cell by a culture solution composition. a to b correspond to DMEM-B medium, and c to d correspond to spermatogonial stem cells in DMEM-C medium (a, c: 100X, c, d: 200X). It is the graph which compared the colony formation number of the chicken spermatogonial stem cell by each growth factor (*: P <0.05). It is a graph which shows the change of the number of chicken spermatogonial stem cells by the combination of a growth factor (*: P <0.001). It is a graph which shows the change of the chicken spermatogonial stem cell number by culture | cultivation temperature. It is a graph applicable to the growth curve of a chicken spermatogonial stem cell at the time of in vitro culture. It is a photograph which shows the culture | cultivation aspect of a chicken spermatogonial stem cell (200X). (a) 3 days of initial culture, (b) 7 days of initial culture, (c) secondary culture (passage 1), 5 days, (d) spermatogonial stem cells cultured for about 3 months (after passage 6, 20 (Day culture) It is a photograph which shows the PAS (Periodic Acid Shiff's) dyeing | staining aspect of the chicken spermatogonial stem cell cultured in vitro (200X). (a) Spermatogonial stem cells (passage 2) of 4-week-old chickens, (b) Spermatogonial stem cells (passage 2) of 9-week-old chickens. It is a photograph which shows the FITC-STA dyeing | staining aspect of the chicken spermatogonial stem cell cultured in vitro (400X). As a result of FITC-STA reaction using spermatogonial stem cells (passage 2) of a 3-week-old chicken, STA strongly reacts on the cell surface and emits fluorescence. (a): Fluorescent photograph, (b): Phase contrast micrograph. It is a photograph which shows the FITC-DBA dyeing | staining aspect of the chicken spermatogonial stem cell cultured in vitro. (a) and (b) are for passage 0 chicken spermatogonial stem cells, and (c) and (d) are for passage 3 chicken spermatogonial stem cells. (a) and (c) are fluorescence photographs, and (b) and (d) are phase contrast micrographs. It is a photograph which shows the reaction mode with respect to the alpha6-integrin antibody of the chicken spermatogonial stem cell cultured in vitro (200X). 3 weeks old chicken spermatogonial stem cells (passage 1). It is a photograph which shows the reaction mode with respect to the β1-integrin antibody of the chicken spermatogonial stem cell cultured in vitro (200X). 3 weeks old chicken spermatogonial stem cells (passage 1). It is a photograph of the SSEA-1, SSEA-3, and SSEA-4 immunostaining results of chicken spermatogonial stem cells cultured in vitro. P indicates the number of passages, and TSC indicates chicken testis matrix cells. It is a photograph of the result of double-staining chicken spermatogonial stem cells cultured in vitro with FITC-STA and anti-SSEA-1 antibody.

Claims (19)

A method for long-term culture of avian spermatogonial stem cells including the following steps:
(a) obtaining avian testis;
(b) separating testicular cell population from the testis; and
(c) culturing spermatogonial stem cells contained in the testicular cell population on a basal cell in a medium containing a cell growth factor.
The method according to claim 1, wherein the step (b) is performed by treating collagenase, trypsin, or a mixture thereof with the obtained testicular tissue.
3. The method according to claim 2, wherein the step (b) is performed by treating the obtained testicular tissue with a mixture of collagenase and trypsin.
The method according to claim 1, wherein in the step (c), the basal cell is a fibroblast, a genital matrix cell, a testicular matrix cell, or a mouse STO cell line.
The method according to claim 4, wherein the basal cell is a genital matrix cell or a testicular matrix cell.
6. The method according to claim 5, wherein the basal cells are genital matrix cells.
The cell growth factor is selected from the group consisting of fibroblast growth factor, insulin-like growth factor-1, stem cell factor, glial-derived neurotrophic factor, and combinations thereof. The method described in 1.
The method according to claim 1, wherein the culture medium further contains a differentiation inhibitor.
The method according to claim 8, wherein the differentiation inhibiting factor is a leukemia inhibiting factor.
The method of claim 1, wherein the medium contains a supplement comprising a mixture of fibroblast growth factor, insulin-like growth factor-1, and leukemia inhibitory factor.
The method of claim 1, wherein the medium further comprises serum and an antioxidant.
The method according to claim 1, wherein the culture temperature in step (c) is about 37 ° C.
The method of claim 1, wherein the birds are chickens, eagle, turkey, duck, eagle bird, eagle, or pigeon.
The method of claim 1, further comprising the step of identifying avian spermatogonial stem cells after the step (c).
The avian spermatogonial stem cells are identified by (i) PAS (Periodic Acid Shiff's) staining, (ii) STA (Sojanum tuberosum agglutinin) staining, (iii) α6-integrin antibody staining, (iv) β1-integrin antibody staining, v) SSEA-1 antibody staining, (vi) SSEA-3 antibody staining, (vii) SSEA-4 antibody staining, (viii) DBA (Doliclos bifflrus agglutinin) staining, or (ix) a combination of the above staining methods. 15. A method according to claim 14, characterized.
A population of avian spermatogonial stem cells characterized by comprising avian cells exhibiting the characteristics of spermatogonial stem cells.
The characteristics of the spermatogonial stem cells are (i) PAS (Periodic Acid Shiff's) staining, (ii) STA (Sojanum tuberosum agglutinin) staining, (iii) α6-integrin antibody staining, (iv) β1-integrin antibody staining, (v This is a positive reaction of SSEA-1 antibody staining, (vi) SSEA-3 antibody staining, (vii) SSEA-4 antibody staining, (viii) DBA (Doliclos bifflrus agglutinin) staining, or (ix) a combination of the above staining methods. A population of avian spermatogonial stem cells according to claim 16, characterized in that.
The population of avian spermatogonial stem cells according to claim 16, wherein the population of avian spermatogonial stem cells is obtained by the method according to claim 1.
Methods for producing transformed birds including the following steps:
(a) transferring a foreign gene to the population of avian spermatogonial stem cells according to any one of claims 16 to 18;
(b) transplanting the avian spermatogonial stem cell population into the testis of the receptor; and
(c) obtaining progeny of the receptor to produce transformed birds;

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